We propose to continue our investigation of the molecular mechanisms by which viruses capture the replication machinery of the host cell and direct this machinery to function solely on the invading viral chromosome. The bacteriophage gamma-Escherichia coli interaction has been chosen as a model system for study of this problem. This is an attractive choice, since the capture and initiation reactions have been reconstituted in vitro in a system composed solely of purified proteins. Biochemical, electron microscopic and immunological methods have been used to demonstrate that an ordered series of protein assembly reactions occurs at the phage replication origin prior to the initiation of DNA replication. For DNA initiation to occur, however, the complete prepriming nucleoprotein structure formed in the assembly reaction must be partially disassembled in an ATP-dependent reaction mediated by the DnaK, DnaJ and GrpE heat shock proteins. The DnaK protein is the E. coli hsp70 homologue and it has been highly conserved over a billion years of evolution. The DnaJ protein is also conserved. These findings and other findings indicate that the hsp70 family of proteins plays vital roles in the physiology of both normal and stressed cells. Hsp70 proteins in eukaryotic cells also function in ATP- dependent protein disassembly reactions and in the translocation of prefolded proteins across intracellular membrane barriers. Little is known, however, about how hsp70 proteins accomplish these feats. The focus of this application is to explore the molecular roles of the E. coli DnaK protein in its interaction with protein and peptide substrates. Specific emphasis will be placed on its interactions with the proteins involved in the initiation of gamma DNA replication and in determining how the DnaJ and GrpE heat shock proteins assist DnaK function. We will explore the interactions of DnaK with short peptides and characterize the peptide-dependent ATPase activity of DnaK. We will examine kinetically the effects of DnaJ and GrpE on this ATPase. Using crosslinking agents, we will initiate studies aimed at localizing the peptide binding site on the DnaK polypeptide. The effect of the GrpE heat shock protein on the assembly and disassembly of preinitiation complexes formed at the phage gamma replication origin will be assessed. The polypeptide stoichiometries and protein-protein interactions in these complex prepriming nucleoprotein structures will be determined. The nature of the product released following the heat shock protein-mediated disassembly reactions will be investigated. We will initiate studies aimed at localizing the functional domains in the DnaK, DnaJ and GrpE heat shock proteins. GroP and DnaB mutant proteins appear to have defects in the assembly of preinitiation complexes at the gamma origin. We will isolate a variety of such mutant proteins and determine at which point in the prepriming pathway the defect is manifested. Studies of the behavior of such mutants should enhance our understanding of the complex protein- protein interactions that underlie the process of initiation of gamma DNA replication as well as the action of the bacterial heat shock proteins.